miR-186上调Dicer1抑制结肠癌细胞侵袭转移的分子机制

为探讨miR-186通过Dicer1影响结肠癌细胞侵袭转移及其作用机制,RT-qPCR检测miR-186在结肠癌组织及正常癌旁组织、人结肠癌细胞及正常人肠上皮细胞HIEC中的表达;荧光显微镜观察稳定过表达/下调miR-186细胞系GFP荧光并采用RT-qPCR检测miR-186表达效率;Transwell及划痕实验检测miR-186对结肠癌细胞SW620和HT29侵袭、迁移能力的影响;免疫荧光法检测上皮间质转化(epithelial mesenchymal transition, EMT)相关分子N-cadherin表达;生物信息学预测及双荧光素酶报告基因实验验证miR-186和Dicer1的靶向关系;免疫组织化学法检测Dicer1在结肠癌组织及正常癌旁组织中的表达;Western blotting检测Dicer1在各细胞株中的表达。结果显示,肿瘤组织中miR-186的表达水平显著低于正常癌旁组织(P<0.05);miR-186在肿瘤细胞中的表达水平显著低于HIEC(P<0.05)。转染过表达/下调miR-186慢病毒阳性质粒及阴性对照的SW620和HT29细胞均出现大量绿色荧光;RT-qPCR显示稳定过表达细胞SW620-mimic-miR-186中miR-186水平显著升高,HT29-inhibitor-miR-186中miR-186水平显著下降,分别与SW620和SW620-mimic-NC、HT29和HT29-inhibitor-NC相比差异有统计学意义(P<0.05)。过表达miR-186能显著降低SW620的侵袭迁移能力,同时抑制N-cadherin水平,而下调miR-186能显著增加HT29的侵袭迁移能力。经www.microRNA.org网站预测发现miR-186和Dicer1有互补结合序列,双荧光素酶报告实验证实两者的靶向结合关系。SW620中Dicer1表达水平显著低于HIEC(P<0.05);稳定过表达细胞系SW620-mimic-miR-186中Dicer1表达水平明显升高,分别与SW620和SW620-mimic-NC相比差异有统计学意义(P<0.05)。由此,miR-186通过靶向正调控Dicer1的表达降低N-cadherin水平从而抑制EMT进程,最终抑制结肠癌细胞的侵袭迁移能力。

miR-186 mediated up-regulation of Dicer1 inhibits colon cancer invasion and metastasis

To study if miR-186 affects invasion and metastasis of colon cancer cells through Dicer1 and its mechanism, the miR-186 expression in colon cancer tissues and normal paracancerous tissues, human colon cancer cell lines and normal human intestinal epithelial cell HIEC were examined by RT-qPCR. Fluorescence microscopy was used to observe GFP fluorescence of stable overexpression/downregulation cell lines and RT-qPCR was used to detect miR-186 expression efficiency. The effect of miR-186 on the invasive ability of colon cancer cells SW620 and HT29 was detected by Transwell assay. Scratch test was used to detect the effect of miR-186 on its migration ability. Immunofluorescence assay was used to detect the expression of N-cadherin, a molecule related to epithelial mesenchymal transition(EMT). Bioinformatics prediction and double luciferase reporter gene experiment were carried out to verify the targeting relationship between miR-186 and Dicer1. Immunohistochemical method was used to detect the expression of Dicer1 in colon cancer tissues and normal adjacent tissues. Western blotting was used to detect the expression of Dicer1 in various cell lines. The results showed that the expression level of miR-186 in cancer tissues was significantly lower than that in normal adjacent tissues(P < 0.05). miR-186 expression level in cancer cells was significantly lower than that in HIEC(P < 0.05). Overexpression/down-regulation of miR-186 or negative control SW620 and HT29 showed strong greeh fluorescence;RT-qPCR showed that the miR-186 level in the stable overexpression cell line SW620-mimic-miR-186 was significantly increased, and in HT29-inhibitor-miR-186 was significantly decreased, differing significantly from SW620 and SW620-mimic-NC, HT29 and HT29-inhibitor-NC, respectively(P < 0.05). Overexpression of miR-186 significantly down-regulated the invasion and migration ability of SW620 and inhibited the level of N-cadherin, while down-regulation of miR-186 significantly increased the invasion and migration ability of HT29. According to the prediction of www.microRNA.org website, miR-186 and Dicer1 had complementary binding sequences, and double luciferase reporting experiments confirmed the targeted binding relationship between them. The expression level of Dicer1 in SW620 was significantly lower than that of HIEC(P < 0.05). The expression level of Dicer1 in SW620-mimc-miR-186 was significantly increased, which was significantly different from that of SW620 and SW620-mimc-NC respectively(P < 0.05). Therefore, miR-186 positively regulates the expression of Dicer1 through targeted binding, and further inhibits EMT process by reducing the level of N-cadherin, leading to the inhibition of invasion and migration ability of colon cancer cells.